While considerable structural diversity is found among drugs and probe molecules, the physical properties of most of these agents with intracellular targets are restricted to a narrow range to ensure transport through the polar extra-cellular milieu and the non-polar lipid bilayer of the cell. Agents falling outside of this range must be tuned often through iterative analogue synthesis to achieve the optimum balance of water solubility and passive membrane transport. A promising approach directed at improving or enabling the cellular uptake of drugs, drug candidates, or probe molecules possessing a wider range of physical properties involves the use of peptide-based molecular transporters to carry these agents actively into cells. See Wender et al., Proc. Natl. Acad. Sci. USA 97:13003–13008 (2000); Mitchell et al., J. Peptide Res. 55:318–325 (2000); Prochiantz, Curr. Opin. Cell Biol. 12:400–406 (2000); Lindgren et al., Trends Pharmacol. Sci. 21:99–102 (2000); Schwartz et al., Curr. Opin. Mol. Ther. 2:162–167 (2000); Schwarze et al., Trends Pharmacol Sci., 21, 45–48 (2000); and Schwarze et al., Trends Cell Biol. 10:290–295 (2000). Representative of this approach, homooligomers (7–9 mers) of L-arginine upon conjugation to various probe molecules (e.g., fluorescein) or drugs (e.g., cyclosporin A) provide highly water soluble conjugates that rapidly enter cells (e.g., human Jurkat). See Wender et al., Proc. Natl. Acad. Sci. USA 97:13003–13008 (2000) and Mitchell et al., J. Peptide Res. 55:318–325 (2000). In addition, drug conjugates of these arginine transporters have been shown to exhibit significant penetration into human skin and to release their drug cargo in targeted T cells (Rothbard et al., Nature Medicine 6:1253–1257 (2000)).
The enormous potential of arginine based molecular transporters is limited for several applications mainly by their availability and cost. Such homooligopeptides are usually prepared using solid-phase peptide synthesis (e.g., Merrifield, J. Am. Chem. Soc. 85:2149–2154 (1963); Atherton et al., Solid-Phase Peptide Synthesis; IRL: Oxford, Engl. (1989); and Fields et al., Int. J. Pept. Prot. Res. 35:161–214 (1990)). Although this approach is readily automated and allows for the synthesis and purification of long peptides, it suffers drawbacks including high cost, limited scalability, and the need for resin attachment and cleavage. In contrast, solution phase synthesis avoids the cost and scale restrictions of resins and in the particular case of oligomers can be conducted using a step-saving bidirectional strategy. Illustrative of the latter point, the uni-directional synthesis of an octamer employing solid phase synthesis requires 14 steps (one coupling and deprotection step for each added monomer), whereas a solution phase bi-directional synthesis of the same octamer would require only seven steps (three coupling and four deprotection steps). See, for example, Appella et al., J. Am. Chem. Soc. 121:7574–7581 (1999); Hungerford et al., J. Chem. Soc., Perkin Trans. I, 3666–3679 (2000); and Chakraborty et al., Tetrahedron Lett. 41:8167–8171 (2000). In the specific case of arginine based peptides, solution phase synthesis offers the additional advantage of avoiding the use of expensive protecting groups for the guanidinium subunit (e.g., Mtr, Pmc and Pbf; see, respectively, Atherton et al., J. Chem Soc. Chem. Commun., 1062–1063 (1983); Ramage et al., Tetrahedron 47:6353–6370 (1991); and Carpino et al., Tetrahedron Lett. 34:7829–7832 (1993)) required in solid phase synthesis.
However, in spite of the advances in the art, there remains a need for a method for the preparation of arginine oligomers, or more generally oligoguanidines that is both cost effective and scalable. The present invention addresses that need.